Fujikawa Mayu, Kobayashi Kazuo, Kozawa Takahiro
The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan
J Biochem. 2015 May;157(5):389-97. doi: 10.1093/jb/mvu085. Epub 2014 Dec 16.
The [2Fe-2S] transcriptional factor SoxR, a member of the MerR family, is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters and functions as a sensor of oxidative stress in Escherichia coli. In the oxidized state, distortion of the target DNA promoter region initiates transcription by RNA polymerase, thereby activating transcription. The inactive reduced state of the protein has remained uncharacterized. Here, we directly observed redox-dependent conformational changes in the promoter DNA by site-specifically replacing selected adenine (A) and cytosine (C) bases in the promoter oligonucleotide with the fluorescent probes 2-aminopurine (2Ap) and pyrrolocytosine (pyrrolo-dC), respectively. Reduction of the [2Fe-2S] cluster in the SoxR-DNA complex dramatically weakened the fluorescence intensity of the 2Ap moieties incorporated into the central part of the DNA. In contrast, the fluorescence of 2Ap moieties incorporated at A in other regions and the fluorescence of pyrrolo-dC moieties in the central region of the DNA (C3 and C3') were only slightly decreased by the reduction. These results strongly suggest that the redox change causes a large conformational change within a region confined to the central A-T base pairs in the promoter region of the DNA.
[2Fe-2S]转录因子SoxR是MerR家族的一员，受[2Fe-2S]簇的可逆氧化还原调节，在大肠杆菌中作为氧化应激的传感器发挥作用。在氧化状态下，靶DNA启动子区域的扭曲会引发RNA聚合酶的转录，从而激活转录。该蛋白的无活性还原状态尚未得到表征。在这里，我们通过分别用荧光探针2-氨基嘌呤（2Ap）和吡咯胞嘧啶（pyrrolo-dC）位点特异性替换启动子寡核苷酸中选定的腺嘌呤（A）和胞嘧啶（C）碱基，直接观察了启动子DNA中依赖氧化还原的构象变化。SoxR-DNA复合物中[2Fe-2S]簇的还原显著减弱了掺入DNA中部的2Ap部分的荧光强度。相比之下，还原作用仅使掺入其他区域A处的2Ap部分的荧光以及DNA中部区域（C3和C3'）的吡咯胞嘧啶部分的荧光略有降低。这些结果有力地表明，氧化还原变化会在DNA启动子区域中央A-T碱基对限定的区域内引起较大的构象变化。
